Denture or removable dental prosthetic made from composite material

ABSTRACT

A curable composite material containing nanodiamonds, denture bases and other dental prosthetics made from the composite material and methods for treating dental stomatitis using these prosthetics.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional of U.S. application Ser. No.18/052,974, now allowed, having a filing date of Nov. 7, 2022 which is aDivisional of U.S. application Ser. No. 17/078,756, now U.S. Pat. No.11,517,408, having a filing date of Oct. 23, 2020, which claims benefitof priority to U.S. Provisional Application No. 62/925,014, having afiling date of Oct. 23, 2019, the entire contents of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The invention pertains to the fields of dental and oral medicine,microbiology, and to the use of nanodiamonds to treat or prevent denturestomatitis associated with Candida albicans and other yeasts.

Description of Related Art

Edentulousness increases in old age and in such cases, a conventionalcomplete denture is commonly the treatment of choice; Fouda S M, et al.,Missing teeth and prosthetic treatment in patients treated at College ofDentistry, University of Dammam. Int J Dent. 2017. Denture bases areconstructed from metal and/or acrylic resin. Acrylic resin, however, ismore frequently used due to its ease of construction and repair,aesthetics and low cost, despite the material's drawbacks of highsurface roughness and low strength; Nandal S, et al., New era in denturebase resins: A review. Dental Journal of Advance Studies. 2013 December;1(03):136-43.

Denture stomatitis (DS) affects more than 70% of patients wearingcomplete dentures; Gendreau L, et al., Epidemiology and etiology ofdenture stomatitis. J Prosthodont. 2011 June; 20(4):251-60. Manyfactors, such as poor oral hygiene; poor-fitting dentures; rough, porousdenture surfaces and systemic diseases, are associated with DS, of whichCandida albicans is considered the main causative pathogen. Thehydrophobicity and surface roughness of denture bases affect the primaryattachment and colonization of Candida albicans; Gendreau L, et al., id;Pereira T, et al., In vitro Candida colonization on acrylic resins anddenture liners: influence of surface free energy, roughness, saliva, andadhering bacteria. Int J Prosthodont. 2007 May 1; 20(3).

Conventional ways to reduce the incidence of DS include mechanicalcleansing, chemical disinfection, special coatings, and/or incorporatingantimicrobial agents in the denture base material; Izumida F E, et al.In vitro evaluation of adherence of Candida albicans, Candida glabrata,and Streptococcus mutans to an acrylic resin modified by experimentalcoatings. Biofouling. 2014 May 28; 30(5):525-33; Ali A A, et al.,Effectiveness of coating acrylic resin dentures on preventing Candidaadhesion. J Prosthodont. 2013 August; 22(6):445-50; Nawasrah A, et al.,Antifungal effect of henna against Candida albicans adhered to acrylicresin as a possible method for prevention of denture stomatitis. Int JEnviron Res Public Health 2016 May 23; 13(5):520; Da Silva F C, et al.Effectiveness of six different disinfectants on removing five microbialspecies and effects on the topographic characteristics of acrylic resin.J Prosthodont. 2008 December; 17(8):627-33.

Conventional cleaning methods are usually effective at eliminatingplaque accumulation from dentures, Da Silva, R C, et al., id. However,performing them may be challenging for elderly patients, particularlythose with physical disabilities or in need of nursing care.

Oral antifungal agents are effective in the treatment of DS, but havetoxic side effects and may lead to the development of resistant strains.In addition, DS recurrence commonly occurs with their use; Garcia-CuestaC, et al., Current treatment of oral candidiasis: A literature review. JClin Exp Dent. 2014; 6:576-582. The antimicrobial effect of chemicaldisinfectants is related to their proper use according to thepreparation guidelines and immersion time; Al-Thobity A M et al., Impactof Denture Cleansing Solution Immersion on Some Properties of DifferentDenture Base Materials: An In Vitro Study. J Prosthodont. 2017; 1-7.

Many studies have investigated the effect of adding antimicrobial orantifungal agents to a denture base resin in an attempt to reducemicrobial and/or fungal adhesion and thereby prevent DS; Sawada T, etal., Self-cleaning effects of acrylic resin containing fluoridatedapatite-coated titanium dioxide. Gerodontology. 2014 March; 31(1):68-75; Nam K Y, et al., Antifungal and physical characteristics ofmodified denture base acrylic incorporated with silver nanoparticles.Gerodontology. 2012; 29(2):e413-19; Li Z, et al., Effect of a denturebase acrylic resin containing silver nanoparticles on Candida albicansadhesion and biofilm formation. Gerodontology. 2016 June; 33(2): 209-16.However, as explained above, the incorporation of antibacterial orantifungal agents can produce toxic side-effects, cause inflammation,disrupt the normal oral biota, or result in development of resistantmicroorganisms especially when used over an extended period of time,such over the lifetime of dentures.

Moreover, the use of these cleaning or disinfection procedures canadversely affect the physical properties of a denture base resin leadingto increased surface roughness, color changes and reduced flexuralstrength; Al Thobity, A M, et al., id; Eg- Porwal A, et al., Effect ofdenture cleansers on color stability, surface roughness, and hardness ofdifferent denture base resins. J Indian Prosthodont Soc 2017; 17:61-67.

Surface roughness (Ra) and hydrophobicity are important properties ofthe denture base material that influence plaque and microbial adhesionand, subsequently, DS; Yamauchi M, et al. In vitro adherence ofmicroorganisms to denture base resin with different surface texture.Dent Mater J. 1990 Jun. 25; 9(1):19-24; Radford D R, et al. Adherence ofCandida albicans to denture base materials with different surfacefinishes. J Dent. 1998 Sep. 1; 26(7):577-83. A rough denture surfaceprovides more area for microbial adhesion. In addition, it protectsentrapped microorganisms from shearing forces during denture cleaning,making their removal difficult even with the use of antimicrobialagents; Pereira-Cenci T, et al. Development of Candida-associateddenture stomatitis: new insights. J Appl Oral Sci 2008; 16(2):86-94;Waltimo T, et al. Adherence of Candida species to newly polymerized andwater-stored denture base polymers. Int J Prosthodont. 2001; 14,457-460.

Denture surfaces with high hydrophobicity have increased adhesion toCandida albicans due to the hydrophobic interaction between the bacteriaand the denture base resin; Waltimo, T. et al., id. Recent attempts toenhance the antimicrobial, mechanical and physical properties ofpolymethylmethacrylate (PMMA) involving the addition of silver, platinumor titanium nanoparticles; has attracted attention because thesemicroparticles enhance the mechanical and physical properties of theresin as well as its antimicrobial resistance. Al Harbi et al., Effectof nanodiamond addition on flexural strength, impact strength, andsurface roughness of PMMA denture base. J Prosthodont. 2019; 28:417-425reported improvement in the mechanical properties of ND-reinforced PMMA.

Several nanosized materials, such as silver, platinum, and titanium,have been added to PMMA, resulting in better resistance to bacterial andfungal colonization; Gad M M, et al., A. PMMA denture base materialenhancement: a review of fiber, filler, and nanofiller addition. Int JNanomedicine 2017; 12:3801; Wang X, et al., Shape-dependentantibacterial activities of Ag ₂ O polyhedral particles. Langmuir. 2009Oct. 9; 26(4):2774-8.

Work has been performed using nanodiamonds in the fields of medicine anddentistry, including guided tissue regeneration, polymer reinforcement,and antibacterial dental implant coatings; Szunerits S, et al.Antibacterial applications of nanodiamonds. Int J Environ Res PublicHealth 2016 Apr. 12; 13(4):413; Najeeb S, et. al. Dental applications ofnanodiamonds. Sci Adv Mater. 2016 Nov. 1; 8(11): 2064-70; Lee D K, et.al. Nanodiamond-gutta percha composite biomaterials for root canaltherapy. ACS nano. 2015 Oct. 15; 9(11):11490-501. NDs possess multiplereactive groups (NH₂, OH) that improve their interfacial bond with PMMA,and are thus considered a compatible filler material; Maitra U, et al.,Mechanical properties of nanodiamond-reinforced polymer-matrixcomposites. Solid State Commun. 2009 Oct. 1; 149(39-40):1693-7; MochalinV N, et al. The properties and applications of nanodiamonds. Nat.Nanotechnol 2012 JaAn; 7(1):11. Although studies have reported on theantimicrobial effect of NDs, none have investigated their effect againstCandida albicans adhesion.

As explained above, denture stomatitis is a significant problem forpeople wearing dentures. Candida albicans plays a significant role inthe morbidity of DS. Accordingly, the inventors sought to develop a wayto reduce the severity of DS by identifying materials that prevent theadhesion of Candida albicans to dentures and other removable dentalprostheses.

BRIEF SUMMARY OF THE INVENTION

As disclosed herein, the inventors have produced a dental compositematerial containing nanodiamonds that inhibits the attachment of yeastssuch as Candida albicans to dentures and other dental prosthetics.Surprisingly, despite the high surface area of nanodiamond particles,the nanodiamonds increased the smoothness of the dental material whilenot substantially increasing its hydrophobicity as determined byscanning electron microscopy and by water contact angle.

The incorporation of nanodiamonds, particularly at low concentrations,reduces Candida adhesion and improves surface roughness.

Aspects of the invention include a method for preventing or treatingdental stomatitis by providing a dental composite material resistant toattachment of the Candida albicans yeast which provides resistance todental stomatitis.

The foregoing paragraphs have been provided by way of generalintroduction, and are not intended to limit the scope of the followingclaims. The described embodiments, together with further advantages,will be best understood by reference to the following detaileddescription taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the disclosure and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by reference to the following detailed description whenconsidered in connection with the accompanying drawings below.

FIGS. 1A-1D. Representative surface images of: Unmodified specimens(FIG. 1A), 0.5% ND concentration (FIG. 1B); 1% ND concentration (FIG.1C); 1.5% ND concentration (FIG. 1D).

FIG. 2 . Representative SEM images of the tested specimens' surfaces.Unmodified specimens (FIG. 2A); 0.5% ND concentration (FIG. 2B); 1% NDconcentration (FIG. 2C); 1.5% ND concentration (FIG. 2D).

FIG. 3 shows PMMA-ND specimens containing 0, 0.5, 1.0 and 1.5 wt %nanodiamonds.

FIG. 4 shows a micrograph of ND-PMMA mixture.

DETAILED DESCRIPTION OF THE INVENTION

One embodiment of the invention is directed to a method for reducing theseverity of denture stomatitis comprising providing a subject in needthereof a prosthetic comprising a composite material incorporatingnanodiamonds. Typically, the subject will have been previously fittedwith dentures to which yeasts such as Candida albicans attach and induceone or more symptoms of denture stomatitis or will be at risk ofdeveloping denture stomatitis, for example, an at-risk subject may havehigh blood sugar, eat a high carbohydrate diet, have diabetes, havecancer or leukemia, HIV/AIDS, be immunosuppressed, have xerostomia, be asmoker, or be on a drug such as an antibiotic, corticosteroid, orimmunosuppressive drug, that can affect the natural microbial flora orimpair or perturb the subject's immune system.

Candida albicans or other yeasts such as C. tropicalis, C. glabrata, C.rugosa, C. parapsilosi, C. dubliniensis, or C. auris may cause primarydenture stomatitis as well as other secondary infections, such as to themouth, throat and esophagus.

DS may be classified as type 1 (localized inflammation or pinpointhyperemia), type 2 (diffuse erythema involving part or all of the mucosain contact with the denture) or type 3 (exhibiting an inflammatorynodular/papillary hyperplasia which may be on the central hard palate oralveolar ridge).

Symptoms of secondary infections include thrush, white patches on theinner cheeks, tongue, roof of the mouth or throat, redness or sorenessin the mouth, throat or esophagus, a cotton-like feeling in the mouth,loss of taste, pain while eating or swallowing or cracking or redness atthe corners of the mouth. Thus, a subject with primary symptoms of DS orsecondary symptoms of yeast infection may be selected for fitting with adenture or dental prosthetic containing nanodiamonds as disclosedherein. In a typical embodiment, a subject will have been fitted withdentures and have one or more symptoms of DS caused by attachment ofCandida albicans to the denture.

A subject may be male or female, old or young, who has been fitted withor other dental prosthetics associated with stomatitis or a yeastinfection, for example, a subject with dentures may be up to 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or >100 years old. Afemale subject may be pregnant or may be undergoing fertility treatmentswith drugs that reduce susceptibility to yeast infections.

The method disclosed herein may be used to treat a subject havingxerostomia, periodontal disease, diabetes, or be infected with humanimmunodeficiency virus (HIV) or is immunosuppres sed.

In the method disclosed herein, the composite material comprises apolymer suitable for use as a denture base, such aspolymethylmethacrylate (“PMMA”) and may be a heat-cured or light-curedpolymethylmethacrylate (“PMMA”). In some embodiments the polymer is PMMAand has a molecular weight as measured by gel permeation chromatograph(GPC) of 50,000, 100,000, 200,000, 500,000, 1,000,000, 1,200,000,1,500,000, 2,000,000, 2,100,000, 2,200,000, 2,500,000 to 3,000,000 orany intermediate value or subrange, for example, from 300,000 to2,000,000. Poly(methyl methacrylate) used widely for prostheticpurposes, it is an amorphous, translucent, thermoset polymer with achemical formula (C₅O₂H₈)_(x) formed by the polymerization of MMAmonomer. The molecular of PMMA affects polymer properties such astensile strength, impact strength, fracture toughness and fatigueresistance. PMMA showed optimum mechanical properties with averagemolecular weight of 10⁵; see Huggett R, Bates J F, Packham D E. Theeffect of the curing cycle upon the molecular weight and properties ofdenture base materials. Dent Mater. 1987 June; 3(3):107-12 (incorporatedby reference)

In the method disclosed herein, the composite material comprisesnanodiamonds have an average diameter ranging from <10, 10, 20, 30, 40,50, 60, 70, 80, 90, 100 or >100 nm in average particle size. Preferably,the average particle size of the nanodiamonds ranges from 10, 20, 30,40, to 50 nm and more preferably about 20 to 40 nm.

The composite material may contain <0.1, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 20 or >2wt. % of the nanodiamonds based on the total weight of the compositematerial. In some preferred embodiments, the composite material willcontain about 0.5 to about 1.5 wt. % nanodiamonds.

In some embodiments, the nanodiamonds may be surface functionalized, forexample, by arylation of oxidized or carboxylated nanodiamonds usingphenylphosponate. In one example, for ND functionalization, ND particleswere heat treated at 450° C. for 2 hours in air to produce functionalgroups on their surfaces; see Avazkonandeh-Gharavol M H, Sajjadi S A,Zebarjad S M, et al: Effect of heat treatment of nanodiamonds on thescratch behavior of polyacrylic/nanodiamond nanocomposite clear coats.Prog Organic Coatings 2013; 76:1258-1264 (incorporated by reference). Anoticeable color change was found with high percent of ND (1.5%) whileless or no color change was noticed with 0.5% ND.

In some embodiments, the method will involve further incorporation ofone or more antimicrobial agents into the composite material along withnanodiamonds. These include plant oils and biological materials (e.g.,rosemary oil, copaiba oil, propolis, shiitake powder, chitosan),quaternized ammonium monomers, metal or metal oxide nanoparticlesincluding silver nanoparticles or titanium oxide nanoparticles,hydroxyapatite, nanoclay, carbon nanotubes or graphenes, silsesquioxaneparticles, and antibiotics or antifungal drugs. In other embodiments,the material comprises, consists essentially of, or consists of, PMMAand nanodiamonds without additional antimicrobial or antifungal agentsor additional nanoparticles.

In one embodiment the composite material contains at least one naturaloil selected from the group consisting of rosemary oil, copaiba oil,peppermint oil, tea tree oil, sage oil, myrrh oil, clove oil, andeucalyptus oil in an amount of 0.05-10 wt %, preferably 0.1-5 wt %,0.25-3 wt %, 0.5-2 wt %, 0.75-1.5 wt %, or 1.0-1.25 wt %, based on thetotal weight of the composite material.

The method as disclosed herein may use a composite material containingnanodiamonds which reduces the numbers of adherent Candida albicans tono more than <100, 95, 90, 80, 70, 60, 50, 40, 30, 20, or 10% of thoseattaching to an otherwise identical composite material not containingnanodiamonds.

Another embodiment of the invention is directed to a composite materialsuitable for use in dentures or dental implants comprising, consistingessentially of, or consisting of PMMA and nanodiamonds and, in someinstances, any inevitable impurities produced during polymerization,mixing or other formulation steps for producing the composite material.In some embodiments, the PMMA is heat-cured, self-cured,radiation-cured, illumination-cured, or otherwise rendered solid by anyother method. In other embodiments, the composite material may compriseother materials having sufficiently low porosity so as to be hygienicfor extended placement in a wearer's mouth. These include plastics andother polymers such as acrylics or methacrylics.

In some embodiments, the composite material will contain nanodiamondshaving an average particle size ranging from about 5 to 100 nm,preferably ranging from about 20, 25, 30, 35, or 40 nm in particle size.

In some embodiments, the composite material comprises nanodiamondshaving an average diameter ranging from <10, 10, 20, 30, 40, 50, 60, 70,80, 90, 100 or >100 nm in average particle size. Preferably, the averageparticle size of the nanodiamonds ranges from 10, 20, 30, 40, to 50 nmand more preferably about 20 to 40 nm. However, a nanodiamond particlesize may be selected which provides a smooth surface to the producedcomposite material, for example, by filling and thus smoothing pores inthe composite material. Preferably, the composite material containingnanodiamonds has a surface roughness Ra of no more than 0.05, 0.06,0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18,0.19, or 0.2 μm. Typically, the composite material containingnanodiamonds as disclosed herein will have a surface roughness less thanthat an otherwise similar composite material not containing thenanodiamonds or which contains equivalent amounts (wt. %) of metal ormetal oxide nanoparticles, ceramic or glass nanoparticles, or carbonnanotubes.

Preferably, the nanodiamonds will be uniformly distributed within thecomposite material or uniformly distributed on the surface of thecomposite material. For example, in some embodiments, the wt % amount ofthe ND component between different unit volumes (e.g., mm³ or cm³) ofthe composite will not vary by more than 1, 2, 5, 10, 15, 20 or 25% orany intermediate value within this range.

In some embodiments the ND component will have a polydispersity index(Mw/Mn) ranging from 0.5, 0.6, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, <1 or 1wherein 1 represents monodisperse ND particles. Distribution of ND mixedwith acrylic powder can be analyzed using SEM. A polydispersity range ofND particles/sheets can be estimated from TEM images, wherein thediameter of nearly spherical sheets was determined to be as large as 150nm and as small as 10 nm, e.g., ranging from about 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, to about 150 nm. In oneexample, to ensure a homogenous and equal distribution of the filler, aND/PMMA mixture was first stirred by glass mortar and pestle and thenwith an electric mixer for an appropriate time. The distribution of theND NPs/sheets within the PMMA matrix was confirmed by SEM examinationwhere ND sheets were thoroughly distributed and some PMMA spheres werecoated with ND (see FIG. 4 , orange arrows).

The composite material incorporating nanodiamonds as disclosed herein istypically used to produce dentures or other dental prosthetics, but insome embodiments, may be used in other devices, such as implants or bonegrafts, or in insertable medical devices which may come into contactwith yeasts or which require a smoother surface compared to devices madeof a composite material like PMMA not containing the nanodiamonds.

Another embodiment is directed to a partial denture, full denture,orthopedic retainer, oral appliance, or other removable dentalprosthetic comprising the composite material as disclosed herein whichcontains nanodiamonds. Other dental materials including implantmaterial, a prosthetic material, a denture material, a filler, a dentureplate material, a mold repairing material, or an impression material mayincorporate the composite PMMA-ND of the invention.

Another embodiment of the invention is a method for making a denture ordental prosthetic comprising mixing a powder comprising nanodiamondshaving a particle size of about 20 to 40 nm with methacrylic resinpowder in a proportion of 98-99.75 wt. % of the resin powder to 2 to0.25 wt. % nanodiamonds, heat polymerizing the mixture into PMMA,casting a denture base from the polymerized PMMA, and, polishing thedenture base. In some embodiments, the polishing is continued until adesired surface roughness is attained, for example, a Ra of 0.2 μm orless. No difficulty was encountered during polishing PMMA-ND specimenswhich the inventors believe was due to the low percentages of NDs.

PMMA may be produced from methyl methacrylate by emulsionpolymerization, solution polymerization, or bulk polymerization. Radicalinitiation may be used, but anionic polymerization of PMMA can also beperformed. PMMA produced by radical polymerization is atactic andcompletely amorphous. All common molding processes may be used,including injection molding, compression molding, and extrusion toproduce a denture base or other device. High quality PMMA sheets may beproduced by cell casting where polymerization and molding steps occurconcurrently. The strength of the material is higher than molding gradesowing to its extremely high molecular mass. Rubber toughening orincorporation of rubber nanoparticles may be used to increase thetoughness of PMMA and prevent brittleness in response to applied loads.

In some embodiments of the invention a different resin or acrylic resinother than PMMA may be used instead of or in admixture with PMMA. Theseinclude acrylic resin, modified acrylic resins, and composite resins andother suitable materials for dentures into which nanodiamonds may beincorporated. PMMA substitutes include materials used in differentfields of medicine and dentistry, including for guided tissueregeneration, polymer reinforcement, and antibacterial dental implantcoatings, as well as reinforcement of provisional resin of fixedprosthesis and gutta percha used for root canal treatment.

The cured denture part may be polished with pumice and/or a lathe, seefor example Kuhar et al., The effect of polishing technique on thesurface roughness of acrylic denture base resin. JPD. 2005; 93: 76-85incorporated herein by reference, to obtain a Ra of 0.2 μm or less.

EXAMPLE

Materials and methods. Specimens were fabricated at the ProsthodonticsLaboratory and tested at the Research Laboratory, College of Dentistry.Microbiology assays were carried out at the Department of Microbiology,College of Medicine, and SEM at the Electron Microscopy Unit, Institutefor Research and Medical Consultations, Imam Abdulrahman Bin FaisalUniversity.

ND/PMMA composite preparation. The nanodiamond (ND) powder (ShanghaiRichem International Co. Ltd) had an average particle size of 30 nm.Transmission electron microscopy (TEM) was used to assess the surfacemorphological features of the powder, which showed that the powderconsisted of graphite sheets (56% wt.) varying from a few to severaltens of nanometers and NDs (44% wt.) with an estimated particle size byTEM of about 30 nm. NDs were added without further purification. Surfacemorphological features and composition were determined by transmissionelectron microscopy (TEM) which showed that the ND powder consisted ofgraphite sheets (56 wt %) varying from a few to several tens ofnanometers and NDs (44 wt %) had an approximate particle size of 30 nm.

The NDs were weighed with an electronic balance (S-234, Denverinstrument) in concentrations of 0.5, 1, and 1.5 wt. % of the acrylicresin powder. The mix was stirred first with gentle hand pressure usinga conventional mortar and pestle, then with an electric mixer for halfan hour at 400 rpm to ensure equal dispersion of the filler in the resinpowder; see Al-Harbi, et al., J Prosthodont. 2019; 28:417-425,incorporated by reference.

Specimen fabrication. Heat-polymerized acrylic was used to fabricate 120specimens resin (Table 1, Major base 20 resin; Prodotti Dentari SPA).The specimens were divided into four groups according to their NDconcentrations, each including 30 specimens. Metal molds were used tofabricate wax specimens 10×10×3 mm³ in size, then the wax specimens wereinvested in dental stone (Fujirock EP; GC) within flasks (61B Two FlaskCompress; Handler Manufacturing). After the setting of the stone wascomplete, the flasks were placed into a wax elimination machine for fiveminutes to dissolve the wax. Some examples of PMMA-ND composites areshown in

FIG. 3 . The PMMA-ND composite is typically used for a denture basewhich contacts a person's gums.

The resulting mold spaces and all the stone surfaces were coated with aseparating medium (Isolmajor; Major Prodotti Dentari Spa). A porcelainjar was used for mixing the polymer and monomer according to themanufacturer's guidelines.

When the mix reached the dough stage, it was hand-kneaded, then packedand processed in a heat-curing unit (KaVo Elektrotechnisches Werk GmbH,Leutkirch, Germany) for two hours at 74° C., followed by one hour at100° C.

Before deflasking, the flasks were left to cool to room temperature.

The specimens were finished and polished with a tungsten carbide bur (HM79GX-040 HP; Meisinger) with a thin cross cut at 18,000 rpm, followed bya fine-grain cylindrical rubber top bur for the acrylic resin(HM251FX-040-HP; Meisinger); Al-Harbi F A, et al. Effect of nanodiamondaddition on flexural strength, impact strength, and surface roughness ofPMMA denture base. J Prosthodont. 2019; 28:417-425 (incorporated byreference).

To standardize the polishing procedures, definitive polishing on apolishing cloth disc (TexMet C10in, 42-3210, Buehler GmbH) was carriedout with a mechanical polisher (Metaserve 250 grinder-polisher, Buehler)at 100 rpm for five minutes in wet conditions; Al Habi, et al., id.After ultrasonic cleaning, the specimens were incubated for one week at37° C. in distilled water that was changed daily to reduce theaccumulation of residual monomers; Murat S, et al. In Vitro Evaluationof Adhesion of Candida albicans on CAD/CAM PMMA-Based Polymers. JProsthodont. 2019 February; 28(2):e873-e879.

Polishing was done in conventional technique followed for denture basepolishing and to standardize the polishing technique we used amechanical polisher as follow: definitive polishing on a polishing clothdisc (TexMet C10in, 42-3210, Buehler GmbH) was carried out with amechanical polisher (Metaserve 250 grinder/polisher, Buehler) at 100 rpmfor five minutes in wet conditions.

TABLE 1 Composition of heat cured acrylic resin and artificial saliva.Name and Materials manufacturer Composition Artificial A.S. Orthana,Mucin, methyl-4-hydroxy- Saliva Biofac A/S, benzoate, benzalconiumchloride, Kastrup, Denmark ethylenediaminetetraacetic acid (EDTA), H2O2,xylitol, peppermint oil, spearmint oil and mineral salts Major.Base.20Prodotti Dentari SPA, Powder: Methyl methacrylate Resin Moncalieri,Italy; (MMA), polymers, benzoyl Powder lot no. peroxide; Form:Micropearls; 14030P; Liquid Liquid: MMA, ethylene glycol, lot no. 1402VPdimethacrylate, N,N-dimethyle- p-toluidine, benzophenone-3

Surface roughness test. A non-contact optical interferometricprofilometer (Contour Gt-K1 optical profiler; Bruker Nano, Inc., Tucson,AZ) was used to measure the Ra (surface roughness) of the specimens at a0.01 mm resolution. The specimens (approximate area 0.43×0.58 mm) werescanned with a standard camera at 20× at five sites, and the average foreach specimen was calculated. A software package (Vision64, Bruker Nano)was used to analyze the acquired images. Pit characteristics weredetermined, and the Ra value of each specimen was calculated.

Scanning electron microscopy (SEM). The scanning electron microscope(SEM) (FEI, ISPECT S50) was used to examine the specimens' surfacecharacteristics (topography). To avoid the non-conductive property ofthe material, a gold coating was applied with a sputter coating machine(Quorum, Q150R ES, UK). Images were captured at different magnifications(500, 1000, 2000, 5000, and 10 000×) to observe importantcharacteristics of the surface changes with different ND concentrations.

Contact angle measurement. After the surface roughness test, thesurfaces of the specimens were gently dried with air. Droplets ofdistilled water were then applied on the surface of the specimens usingan auto pipette and a goniometer to standardize the droplets volume (2μL). An automated contact angle goniometer (DM-501; Kyowa InterfaceScience Co, Japan) was used to measure the contact angle. The angle ofthe tangent to the surface of the water droplet was measured andrepeated four times on different areas of each specimen. Thereafter, theaverage was calculated. The images were analyzed with FAMAS software(Kyowa Interface Science Co, Japan).

Microbiology test. The specimens were sterilized with 70% alcohol, thencleaned ultrasonically with sterilized distilled water; Mura, et al.(2019), id. The sterilized specimens were soaked in artificial saliva(Table 1) containing 2,000,000 cells of Candida albicans (ATCC 10231) at37° C. for 48 hours; Ali, A A, et al. (2013), id., Nawasrah, A, et al. () 2016), id., Gad M M, et al., Inhibitory effect of zirconium oxidenanoparticles on Candida albicans adhesion to repaired polymethylmethacrylate denture bases and interim removable prostheses: a newapproach for denture stomatitis prevention. Int J Nanomedicine. 2017;12:5409.

To detach non-adherent cells, phosphate-buffered saline (PBS) was usedto wash the acrylic plates three times. The plates were then put intosterile tubes containing 1 ml of Sabouraud's dextrose broth(SDB—Acumedica Co., Manufacturers, Inc.) for 24 hours. After that, theplates were vibrated for 10 minutes with a vortex mixer. To obtainclustered pellets of Candida albicans, the tubes were then centrifugedfor five minutes at 4,500 rpm.

After being centrifuged, the acrylic resin specimens were extracted fromthe tubes, and the clustered pellets were collected from the tube. TheCandida albicans attached to each specimen was counted by two methods:

Slide count method (Neubauer): For microscopic evaluation, 2.5 μl ofTrypan Blue 0.4% solution in phosphate (MP-Biomedicals) was added to 7.5μl of each concentrated Candida pellet of the specimen positioned on aslide worktable (Neubauer Slide Counter; Chambers-Marienfeld). TheTrypan Blue stain distinguishes the living Candida albicans from thedead by showing the living Candida albicans cells as transparent andsurrounded by a blue borderline, while the dead cells were colored blue.A light microscope (at low power magnifications, 10×) was used to countthe number of Candida albicans cells. Each slide contained four mainsquares, each of which was divided into 16 smaller squares. Candidaalbicans cells were counted in two main squares, then multiplied by twoto achieve the total number of Candida albicans on each slide; Ali, A A,et al. (2013), id; Nawasrah, A, et al. (2016), id.

Direct culture method: [colony-forming unit (CFU)] 10 μl of eachisolated centrifuged pellet was spread onto a petri dish and incubatedfor 24 hours at 37° C.; Gad, M M. et al., (2017), id. A marker pencounter (colony counter “SP Scienceware, Bel-Art Products”) was used tocount the Candida albicans colonies. The number of colonies wascalibrated for the dilution factor. When the number of colonies reached5,000 or more, it was considered overgrown.

Statistical analysis. An IBM SPSS Statistics 23 (IBM Corp., Armonk, NY)was used for all statistical analyses. Arithmetic means and standarddeviations for categorized parameters were calculated. ANOVA was used tocheck overall significance, and pairwise significance was tested byusing Tukey's post hoc test. The level of significance was set atP<0.05.

In comparison to the control group, the addition of NDs significantlyreduced the surface roughness (P<0.05) (Table 2). The highest Ra valuewas found with the control group (0.129±0.011 μm), while the lowestvalue was with 0.5% NDs group (0.039±0.009 μm) with no significantdifference between 0.5% NDs and 1% NDs groups (P=0.396).

Significant differences were seen between the 0.5% NDs and 1% NDs groupsand the 1.5% NDs group (P<0.05), which showed the highest Ra value ofthe NDs groups.

FIG. 1 shows the color parameter that represents the average Ra valuesranging from red at the parameter top to blue at the parameter bottom.Where rough surfaces have peaks, valleys, and areas in between, redreveal the peaks while blue reveals the valley depth, and theinterdigitated colors in between exhibited areas between the peaks andvalleys. Thus, the graduated color of this parameter displayed the wholesurface roughness. FIGS. 1A, 1B, 1C and 1D, respectively, depict surfaceroughness for (unmodified specimens, 0.5% nanodiamond (ND)concentration; 1% ND concentration; and 1.5% ND concentration.

FIG. 2 shows SEM micrographs of the surface of the specimens at 5000×magnification with different concentrations of NDs. SEM analysisdisplayed high Ra in unreinforced heat-cured acrylic resin (FIG. 2A)containing broad scattered pores with dimensions of 80 to 100 micronsand depth variations.

FIG. 2B (0.5% NDs) shows a compact morphology with diminutive porespreoccupied by NDs measuring a few microns in diameter.

FIG. 2C (1% NDs) and FIG. 2D (1.5% NDs) show smooth surfaces resultingfrom NDs filling the pores. FIG. 2D also shows loosely attached clustersof NDs on the surface of the specimens.

The mean and standard deviations to surface roughness in these samplesis described by Table 2.

TABLE 2 Mean and standard deviations of surface roughness of studiedgroup Group Control 0.5% ND 1% ND 1.5% ND Mean ± SD 0.129 ± 0.011 0.039± 0.009^(A) 0.047 ± 0.007^(A) 0.102 ± 0.017 F-value (149.089) P-value(0.000)

Table 3 shows the mean values of the contact angles in each groupagainst distilled water. No significant difference was found in contactangles between all the tested groups (p=0.083) or between the controlgroup and the NDs-reinforced groups (p>0.05). The highest contact anglevalue was 84.8±1.62, recorded with the 1.5% NDs group, while the lowestcontact angle value was 82.4±1.8, recorded with the 0.5% NDs group.

TABLE 3 Mean and standard deviations of contact angles in all testedgroups Group Control 0.5% ND 1% ND 1.5% ND Mean ± 82.9 ± 3.14^(A) 82.4 ±1.8^(A) 83 ± 1.63^(A) 84.8 ± 1.62^(A) SD F-value = 11.025 P-value =0.083

Table 4 shows the means and standard deviations of all the tested groupsregarding the Neubauer and CFU tests.

Regarding Neubauer, the Candida albicans count in the control groupshowed a significant increase compared with the other NDs groups(P<0.05), the lowest Candida albicans count occurring in the 1% NDsgroup (276.2±34.47). In the NDs groups, the 0.5% NDs group showed asignificant difference compared with the 1% and 1.5% NDs groups, whilethe 1% NDs and 1.5% NDs groups showed no significant difference betweenthem.

The number of living Candida albicans cells was obtained with a culturetest.

The CFU method revealed a significant increase in the Candida albicanscount in the control group compared to the NDs groups (P<0.05), with thelowest CFU count occurring in the 1% NDs group (2848.6±187.8). In theNDs groups, the 0.5% NDs group showed a significant difference comparedwith the 1% and 1.5% NDs groups, while the 1% and 1.5% NDs groups showedno significant difference between them. The number of Candida albicansobserved with the CFU test decreased significantly with the addition ofNDs, especially in the 1% and 1.5% NDs groups.

The Neubauer and CFU test methods showed no statistically significantdifference, which confirmed the effect of NDs against Candida albicans,as their effect was approximately the same regardless of concentration.

TABLE 4 Mean and standard deviations of different studied groupsregarding slide count (Neubauer) and colony forming unit (CFU). GroupNeubauer CFU Control 2035.9 ± 121.03ª 13212.0 ± 821.2^(A) 0.5 ND 1039.4± 88.11^(b) 7604.7 ± 310.97^(B)   1 ND 276.2 ± 34.47^(c) 2848.6 ±187.8^(C) 1.5 ND 303.3 ± 35.93^(cd) 3602.0 ± 339.25^(CD) F 21.36 104.2 P  0.01*       0.0001*

Lower case letters a, b, c and d indicate slide count tests. Upper caseletters A, B, C and D indicate cell culture counts. In Tables 2-4:F=ANOVA test, One-way ANOVA and Tukey's post hoc test (p<0.05) indicatedifferences among NDs concentrations. Differences are indicated bysuperscripts which are lettered vertically where use of the same letter,e.g., “cd” or “CD” indicates no significant difference.

The results revealed a significant decrease in Candida albicans adhesionwith the addition of NDs in comparison with the control group.

Surprisingly, incorporation of nanodiamonds into the PMMA dental basesignificantly reduced surface roughness compared to the control groupbut the water contact angles were not significantly changed. The firstresearch hypothesis was partly rejected because the surface roughness ofthe NDs-PMMA groups was significantly reduced compared to the controlgroup, while the contact angle values showed no significant change. Thisresult suggests that incorporation of nanodiamonds into PMMA wouldreduce the ability of Candida albicans to attach to the composite dentalcomposite material because Candida albicans attaches better to roughsurfaces with greater surface area for colonization.

The present study showed a significant decrease in the Candida albicanscount in the NDs groups compared with the control group, which had thehighest Ra value. According to the surface roughness test and SEManalysis, the addition of NDs to PMMA improved the surface structure ofthe specimens, which could contribute to the prevention of Candidaalbicans adhesion.

In comparison with the control group, SEM analysis showed that theaddition of NDs changed the surface profile of the tested specimens byfilling the pores. After complete saturation, NDs formed clusters on thesurface of the specimens.

While not being bound to any particular theory, the inventors believethat reduced surface roughness may have resulted from the nanodiamondparticles' small size, which reduced the inter-particle distance,resulting in close contacts between the nanoparticles at lowerconcentrations. While an increase in Ra was observed with a highconcentration of NDs (1.5%), even at this higher concentration of NDs,the surface roughness was still lower than in the control group andwithin a clinically acceptable value of about 0.2 μm; see Radford D R,et al., Adherence of Candida albicans to denture base materials withdifferent surface finishes. J Dent. 1998 Sep. 1; 26(7):577-83. Thisincrease in the sample containing 1.5 wt. % ND, may have resulted fromspaces created on the surface of the specimens due to the separation ofloosely attached clusters of NDs particles after finishing andpolishing. The reduced Ra of PMMA incorporating NDs may have resultedfrom improved polishability due to the presence of small nanofillersmerging within the denture base resin, providing a smooth polishedsurface.

The addition of NDs to PMMA showed no significant change in the contactangles of the specimens containing NDs in comparison to the controlgroup. This result is surprising because disagrees with those of severalstudies that reported changes in the PMMA contact angle with theaddition of nanofillers. Increasing carbon nanotube content of PMMA inthe 1-2 wt % range increased the water contact angle; Kim K I, et al.,Carbon nanotube incorporation in PMMA to prevent microbial adhesion.Scientific reports. 2019 Mar. 20; 9(1):4921. Such an increase wouldenhance adhesion of Candida albicans; Sipahi C, et al. The effect ofacquired salivary pellicle on the surface free energy and wettability ofdifferent denture base materials. J Dent 2001; 29:197-204.

The Neubauer and CFU tests showed a significant decrease in the Candidaalbicans count in the NDs groups compared with the control group. Thelowest Candida albicans count in both tests occurred in the 1% NDsgroup.

The effect of NDs against Candida albicans adhesion has not previouslybeen disclosed Candida albicans is the main causative pathogen ofdenture stomatitis, which affects many complete denture subjects. Asdisclosed herein, the addition of nanodiamonds (NDs) topolymethylmethacrylate (PMMA) denture composite material significantlydecreased surface roughness and Candida albicans adhesion to thecomposite material with the lowest values observed at 1% NDs and 0.5%NDs, respectively, while not significantly affecting contact angle (i.e.not increasing hydrophobicity). These properties are advantageous intreating dental diseases such as dental stomatitis caused by Candida.These results show that PMMA/NDs composites are valuable for preventionof denture stomatitis which is considered one of the most commonclinical problems among removable denture wearers.

Terminology

Terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

It will be further understood that the terms “comprises” and/or“comprising,” when used in this specification, specify the presence ofstated features, steps, operations, elements, and/or components, but donot preclude the presence or addition of one or more other features,steps, operations, elements, components, and/or groups thereof.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items and may be abbreviated as“/”.

As used herein in the specification and claims, including as used in theexamples and unless otherwise expressly specified, all numbers may beread as if prefaced by the word “substantially”, “about” or“approximately,” even if the term does not expressly appear. The phrase“about” or “approximately” may be used when describing magnitude and/orposition to indicate that the value and/or position described is withina reasonable expected range of values and/or positions. For example, anumeric value may have a value that is +/−0.1% of the stated value (orrange of values), +/−1% of the stated value (or range of values), +/−2%of the stated value (or range of values), +/−5% of the stated value (orrange of values), +/−10% of the stated value (or range of values),+/−15% of the stated value (or range of values), +/−20% of the statedvalue (or range of values), etc. Any numerical range recited herein isintended to include all sub-ranges subsumed therein.

Disclosure of values and ranges of values for specific parameters (suchas temperatures, molecular weights, weight percentages, etc.) are notexclusive of other values and ranges of values useful herein. It isenvisioned that two or more specific exemplified values for a givenparameter may define endpoints for a range of values that may be claimedfor the parameter. For example, if Parameter X is exemplified herein tohave value A and also exemplified to have value Z, it is envisioned thatparameter X may have a range of values from about A to about Z.Similarly, it is envisioned that disclosure of two or more ranges ofvalues for a parameter (whether such ranges are nested, overlapping ordistinct) subsume all possible combination of ranges for the value thatmight be claimed using endpoints of the disclosed ranges. For example,if parameter X is exemplified herein to have values in the range of 1-10it also describes subranges for Parameter X including 1-9, 1-8, 1-7,2-9, 2-8, 2-7, 3-9, 3-8, 3-7, 2-8, 3-7, 4-6, or 7-10, 8-10 or 9-10 asmere examples. A range encompasses its endpoints as well as valuesinside of an endpoint, for example, the range 0-5 includes 0, >0, 1, 2,3, 4, <5 and 5.

As used herein, the words “preferred” and “preferably” refer toembodiments of the technology that afford certain benefits, undercertain circumstances. However, other embodiments may also be preferred,under the same or other circumstances. Furthermore, the recitation ofone or more preferred embodiments does not imply that other embodimentsare not useful, and is not intended to exclude other embodiments fromthe scope of the technology. As referred to herein, all compositionalpercentages are by weight of the total composition, unless otherwisespecified. As used herein, the word “include,” and its variants, isintended to be non-limiting, such that recitation of items in a list isnot to the exclusion of other like items that may also be useful in thematerials, compositions, devices, and methods of this technology.Similarly, the terms “can” and “may” and their variants are intended tobe non-limiting, such that recitation that an embodiment can or maycomprise certain elements or features does not exclude other embodimentsof the present invention that do not contain those elements or features.

The description and specific examples, while indicating embodiments ofthe technology, are intended for purposes of illustration only and arenot intended to limit the scope of the technology. Moreover, recitationof multiple embodiments having stated features is not intended toexclude other embodiments having additional features, or otherembodiments incorporating different combinations of the stated features.Specific examples are provided for illustrative purposes of how to makeand use the compositions and methods of this technology and, unlessexplicitly stated otherwise, are not intended to be a representationthat given embodiments of this technology have, or have not, been madeor tested.

The citation of references herein does not constitute an admission thatthose references are prior art or have any relevance to thepatentability of the technology disclosed herein. Any discussion of thecontent of references cited is intended merely to provide a generalsummary of assertions made by the authors of the references, and doesnot constitute an admission as to the accuracy of the content of suchreferences.

The invention claimed is:
 1. A partial denture, full denture, or otherremovable dental prosthetic comprising a composite material, comprising:poly(methylmethacrylate) (PMMA) and 0.1 to 1.0 wt. % nanodiamonds basedon a total weight of the composite material, wherein said nanodiamondshave an average particle size ranging from 20 to 40 nm; wherein the PMMAhas a molecular weight of 50,000 to 1,000,000 g/mol, wherein thecomposite material is porous having pores 80 to 100 μm in size, whereinthe nanodiamonds fill pores of the composite material, where thenanodiamonds are only in the pores on the composite material, andwherein the composite material has a surface roughness (Ra) of no morethan 0.2 μm.
 2. The partial denture, full denture, or other removabledental prosthetic of claim 1, wherein the composite material furthercomprises rubber nanoparticles.
 3. The partial denture, full denture, orother removable dental prosthetic of claim 1, wherein the PMMA has amolecular weight as measured by gel permeation chromatograph (GPC) of300,000 to 1,000,000 g/mol.
 4. The partial denture, full denture, orother removable dental prosthetic of claim 1, wherein the nanodiamondsin the composite material have an average particle size of about 30 nmand the nanodiamonds are uniformly distributed in the composite materialso that the density of nanodiamonds between cm³ different volumes ofcomposite does not vary by more than 10%.
 5. The partial denture, fulldenture, or other removable dental prosthetic of claim 1, comprising0.25 to 1 wt. % nanodiamonds based on a total weight of the compositematerial.
 6. The partial denture, full denture, or other removabledental prosthetic of claim 1, wherein the nanodiamonds have an averagediameter of about 30 nm.
 7. The partial denture, full denture, or otherremovable dental prosthetic of claim 1, wherein the nanodiamonds arepresent in an amount ranging from 0.5 to 1 wt. % of the compositematerial.
 8. The partial denture, full denture, or other removabledental prosthetic of claim 1, wherein Candida albicans binds to saidcomposite material less than a control or otherwise identical compositematerial not containing nanodiamonds.
 9. The partial denture, fulldenture, or other removable dental prosthetic of claim 1, consisting ofthe composite material.
 10. The partial denture, full denture, or otherremovable dental prosthetic of claim 1, wherein the composite materialconsists of the PMMA and the nanodiamonds.
 11. The partial denture, fulldenture, or other removable dental prosthetic of claim 1, which is inthe form of a partial denture.
 12. The partial denture, full denture, orother removable dental prosthetic of claim 1, which is in the form of afull denture.